Although a number of anticancer drugs have been developed in the last 60 years, there is a lack of effective drugs against many solid tumors such as triple-negative breast cancer (TNBC), brain cancer and melanoma. Therefore, there is an urgent need to develop novel anticancer agents against these solid tumors. Superstolides A and B, natural products isolated from marine sponge, exhibit potent anticancer activity. Their unique chemical structure indicates they may likely have a novel cellular target. But their scarcity plus the overwhelming difficulty in developing a practical total synthesis has severely impeded research into a thorough understanding of their mechanism of action and has hampered the gain of further insight into their clinical potential. We have solved the supply problem by synthesizing a truncated superstolide A (named as ZJ-101) that retains the full anticancer activity of the natural product. The cell lines of some of the most common and highly metastatic TNBC, CNS and melanoma are especially sensitive to ZJ-101 (GI50 < 10 nM). In particular, ZJ-101 appears to be most effective against cell lines with p53 mutations, which are known to cause drug resistance. Thus, ZJ-101 represents a new promising anticancer agent with a novel and, as yet, undefined mode of action. Due to its promising anticancer activity ZJ-101 has recently been selected by NCI to undergo additional preclinical evaluation. The objectives of this proposal are to conduct preclinical evaluation to assess the anticancer therapeutic potential of ZJ-101 and investigate its underlying mechanism of action. The central hypothesis of the proposal is that ZJ-101 can serve as an excellent advanced lead compound that biologically mimics its parental natural product, and a collaborative medicinal chemistry and chemical biology investigation of ZJ-101 will lead to the development of new effective anticancer agents and the discovery of novel anticancer drug target(s). The specific aims of the proposal are: 1) to synthesize a sufficient quantity of ZJ-101 for preclinical evaluation and biological investigation. We will prepare 500 mg of ZJ-101 using our efficient synthetic process. The material will be supplied to NCI for hollow fiber test and to the co-PI at Johns Hopkins for biological study. 2) to conduct biological investigations of ZJ-101 and determine its mechanism of action. We will investigate the biochemical/molecular basis responsible for the potent antiproliferative activity of ZJ- 101. A number of clickable photoaffinity-labeled or fluorescent-labeled ZJ-101 probes will be synthesized and used for the identification of its molecular target(s) and elucidation of its mechanism of action. Various biochemical and molecular biology methods will be employed for target validation. Results from this study will be highly significant not only in advancing the development of novel anticancer agents from superstolides by assisting NCI to evaluate the anticancer therapeutic potential of ZJ- 101, but also for gaining fundamental new knowledge in the discovery of new anticancer drug target(s).